Comparison between Chondrogenic Markers of Differentiated Chondrocytes from Adipose Derived Stem Cells and Articular Chondrocytes In Vitro.

OBJECTIVE(S)
Osteoarthritis is one of the most common diseases in middle-aged population in the world. Cartilage tissue engineering (TE) has been presented as an effort to introduce the best combination of cells, biomaterial scaffolds and stimulating growth factors to produce a cartilage tissue similar to the natural articular cartilage. In this study, the chondrogenic potential of adipose derived stem cells (ADSCs) was compared with natural articular chondrocytes cultured in alginate scaffold.


MATERIALS AND METHODS
Human ADSCs were obtained from subcutaneous adipose tissue and human articular chondrocytes from non-weight bearing areas of knee joints. Cells were seeded in 1.5% alginate and cultured in chondrogenic media for three weeks with and without TGFβ3. The genes expression of types II and X collagens was assessed by Real Time PCR and the amount of aggrecan (AGC) and type I collagen measured by ELISA and the content of glycosaminoglycan evaluated by GAG assay.


RESULTS
Our findings showed that type II collagen, GAG and AGC were expressed, in differentiated ADSCs. Meanwhile, they produced a lesser amount of types II and X collagens but more AGC, GAG and type I collagen in comparison with natural chondrocytes (NCs).


CONCLUSION
Further attempt should be carried out to optimize achieving type II collagen in DCs, as much as, natural articular chondrocytes and decline of the production of type I collagen in order to provide efficient hyaline cartilage after chondrogenic induction, prior to the usage of harvested tissues in clinical trials.


Introduction
Based on the current statistics, millions of people in the world suffer from articular diseases which are associated with joint destruction (1)(2)(3). Traditional treatments, despite some successes, have not yet been satisfactory (4,5). Thus tissue engineering which is a combination of engineering principles and biological sciences has been considered to have an important role in the treatment of articular lesions (6). Tissue engi neering is seeking for cellular biomaterial scaffolds and appropriate stimulating growth factors to produce alternative hyaline articular cartilage that could be transpl anted i n order to treat articular defects (7)(8)(9)(10).
The selection of suitable cells is often difficult, as cells should be autologous (not responsive immunologically), having hi gh proliferative power and potentially differentiable to the desired target cells (11). Autologous chondrocytes are the first differentiated cells used for cartilage tissue engineering, then neonatal (12) and finally fetal (13) chondrocytes. Due to some limitations of using mentioned cells, adult undifferenti ated stem cells with high differentiation ability to chondrocy tes, have been considered as an alternative. Among different sources of stem cells, those of adipose derived stem cells (AD SCs) and bone marrow derived mesenchyme s tem cells (BM-M SCs ) have been used in cartilage tissue engineeri ng (15)(16)(17). In recent years due to eas e of accessibility to subcutaneous adi pose tissue and availability of large quantity of cells compared with the bone marrow, ADSCs have received a great interest (18)(19)(20). So far, the characteristics of ADSCs have not been well defined and more i nvestigation is necessary (20,21). Another important factor in TE is selection of proper biomaterial scaffold which should have biocompatibility, biodegradability and facilitator of tissue proliferation and proper biomechanical properties (23,24). In TE both hydrogels and porous scaffolds are used. Alginate is a kind of natural hydrogel which is derived from seaweed. Many researchers have investigated this hydrogel as an injectable seeded chondrocytes scaffold for articular cartilage repairmen (25)(26)(27). Previous studies have indicated that differentiated chondrocytes from ADSCs in alginate were morphologically spherical and similar to natural chondrocytes. Moreover, differentiation trend requires other suitable growth factors as well as scaffolds such as TGFβ, FGF, IGF and BMP (1). Among these, TGFβs including TGFβ1, TGFβ2, TGFβ3 and BMPs have the most potential for differentiation induction to chondrocytes in MSCs. It is reported that TGFβ2, TGFβ3 are more effective in induction of human MSCs to chondrocytes than TGFβ1 (30). In the past two decades except few cases no suitable cartilage tissue has been designed in vitro for clinical applications (14,31). Therefore, it seems that TE is still at its infancy and in order to address many clinical issues, a long way should be followed. It is hoped that, by clarifying ambiguities such as, precise mechanisms of differentiation of stem cells to the chondrocytes, the pattern of interaction with various scaffolds, further determination of differences between articular chondrocytes. Moreover, the differentiated chondrocytes from stem cells, a functional long life engineered cartilage tissue similar to articular cartilage would be presented to the medical fields (1,29).
Many scientists hav e compared chondrogenesis between articular chondrocytes and chondrocytes derived from stem cells. Gleghorn (2010) i ndicated the higher chondrogenic potential in fetal chondrocytes compared wi th ADSCs in polyglycolic acid (PGA) (13). Despite such investigation on differentiation of ADSCs to chondrocytes and articular chondrocytes in alginate, no reported comparison between these cells has been made. Therefore, we aimed to assess and compare types II, X, I collagens, aggrecan and glycosaminoglycan (GAG) of differentiated chondrocytes from AD SCs, wi th natural articular chondrocytes s eeded in alginate with and without TGFβ3 on days 14 and 21.

Materials and Methods
Isolation and proliferation of ADSCs Written consent was obtained from all patients i n an operation room. Subcutaneous adipose tissue (∼10 g) was obtai ned from 4 pati ents (30-50 years age), under s terile condi tions and transferred to the lab. Afterward, it was di gested with 0.075% collagenase type I (Sigma) at 37°C for 30 min. The enzyme was inactivated by DMEM LG (Sigma) contai ning 10% FBS (Invitrogen). Subsequently, the resultant solution was centrifuged (1200 rpm, 15 min) and cell pellet was cultured in 25 cm 2 flasks with DMEM LG, 10% FBS, 1% penicillin & streptomycin (Gibco) and incubated wi th 5% CO2, 37°C. The medium was changed every 4 days. , Cells were detached with 0.05% try psin/0. 53 mM EDTA (Sigma) and passaged, when they reached 80% confluence. P3-p5 cells were seeded in an alginate scaffold.

Isolation& proliferation of articular chondrocytes
After taking a wri tten consent, a bit of articular cartilage was obtained by the knee arthroscopy from 4 patients (20-30 years age) whose joints di d not show any signs of a degenerative arthritis. The specimens were taken from non-weight bearing areas and transferred wi thin PBS (Si gma) to the lab. The specimens were then divided into 1×1 mm slices and diges ted with collagenase type II (Sigma) 350 u/ml in 37° for 4 hr. After inactivation with DM EM F12 (Gibco) with 10% FBS, the solution was centrifuged and cell pellets were cultured in 25 cm 2 flask with DMEM F12, 10% FBS, 1% penicillin and streptomycin. When cells reached 80% confluence, they were detached wi th trypsin/EDTA and passaged. P2-P4 cells were used for seeding in an alginate scaffold.

Biochemical assays DNA quantification
In order to determinate DNA and GAG , fi rst, alginate beads on days 14 and 21 digested for 18 h at 60° in papain solution 125µg/ml(Sigma) containing cistein10 mM (Sigma) in PB E buffer (Na2Hpo4 100 mM, EDTA 10 mM, pH=6.5). For each 12 beads, one ml enzyme was used. The resultant solutions were used for determination of DNA and GAG.
The content of DNA (ng/ml ) was measured using DNA Quantification Kit, Fluorescence Assay (Sigma, Cat. No. DNAQF), according to its manufacturer's protocol. Briefly, res ultant solution was read by dyed Hoechst 33258 and absorbance of each sample was read by spectrofluorometer (Perkin Elmer LS-3) at 360 nm in exci tation and 460 nm wave length in emission. Afterward, the DNA content was calculated according to the thymic calf standard curve.

GAG Assay
The amount of GAG (µg/ml) was quantified using 1, 9-dimethylmethylene blue (DMMB) dye. Briefly, 100μl of res ultant solution was mixed with 2400 μl of DMMB solution in a cuvette and then its absorbance in 525 nm wave l ength was read by spectrophotometer (Spectonic 70, Baucsh&lomb). The GAG content was calculated using standard curve chondroitin sulfate of bovine trachea.
Finally, the ratio of GAG/DNA in each sample was normalized. All experiments were performed twice.

Real-time PCR
At first, alginate beads on days 14 and 21 were washed with PBS and for digestion, they were placed in 1.5% 55 mM citrate sodium (Sharlau) and 0.15 mM NaCl (Merck). The resultant solution centrifuged for 10 mi n at 1200 rpm and the derived cell pellet was used for the extraction of RNA with RNeasy mini kit (Qiagen,Cat. No. 74101) with a little modification (36). For lysing cells, firstly a solution mixed of 990 µl Trizol (Invitrogen) and 10 µl of 2-mercaptoethanol (Sigma), was kept i n room temperature (RT) for 5 min. Next, 200 µl chloroform was added and shacked vigorously for 15 second kept in RT for 2-3 min and then centrifuged for 15 min at 4° at 12000 g.
The supernatant aqueous phase was trans ported into a 1.5 ml microtube and the same volume of ethanol 70% was added and then mixed. The resultant solution transferred to columns in kit and the rest of ins truction was carried out according to kit protocol in which to kit protocol in which RNase free DNase set (Qiagen) applied for elimination of possible DNA contami nation. The extent of derived RNA was measured by spectrophotometer (Biophotometer, Eppendorf) at 260/280 nm wave length. Reverse transcri ption for cDNA, 100 ng RNA used by recruitment of RevertAid TM First Strand cDNA Synthesis Kit (Fermentas,Cat. No. #K 1621) according to manufacturer's protocol.
Relative quantification of the expression types II and X collagens was measured, using M axima SYBR® Green/RoxqPCR mas ter Mix 2X (Fermentas), with GAPDH primer as an internal control. The calculation was performed via comparative Ct (ΔΔ Ct). The reactions conducted in 20 µl with, 10 µl SYBR® Green, 7.5 µl H2o, 0.25 µM forward and rev erse primers and 1.5 µl cDNA as following planned by StepOne Plus Real Time PCR system (Applied Biosystem): primary denaturation in 95° for 10 min, denaturation in 95° for 15 sec, Annealing and Extension in 60° for 1 min -the whole process was done 40 cycles-and finally melt curv e (increment 0.3 °C, 60°C→95°C) was depicted. All experiments were performed in triplicates for each specimen. The applied primers for Real-Time PCR were designed by AlleleID 7/6 software which indicated in Table 1.

Statistical tests
Kolmogorov-Simonov test was used for assessing the normal distribution of vari ables. ANOVA (oneway-analysis of variance) with LSD post hoc test used for comparison of ELISA, Real-Time PCR and GAG assay results in different groups.

Gene expression of type II and X collagens
The results of Real-Time PCR indicated that type II collagen gene expression in articular chondrocytes (NCs) is significantly higher (P<0.001) than differentiated chondrocytes (DCs) after two weeks.  TGFβ3 resulted in a significant (P<0.001) decrease of type II collagen expression in NCs, but its expression reduced strongly in both groups of NCs at the end of third week. Also, TGFβ3 caused increase in type II collagen gene expression in DCs i n third week than in second week. Nev erthel ess, DCs expressed the lowest amount of type II collagen in second and third weeks. (Figure 1, A) ( Table 2). The highest amount of type X collagen gene expression, a hypertrophic factor, was seen in articular chondrocy tes (NCs ) in third week which was significantly (P<0.05) more than differentiated chondrocytes (D Cs), while it was not expressed in thes e cells in the second week. Adding TGFβ3 led to significant increase of gene expression in third week (P<0. 001) compared to NCs without TGFβ3. Also, DCs in the presence of this growth factor significantly (P<0.05) expressed type X collagen higher than other group without it at the end of second week. Generally, with the course of time and in third week D Cs no longer expressed this gene (Figure 1, B) ( Table 2).

Production of Aggrecan, Type I collagen and GAG
The results of ELISA indicated that articular chondrocytes (NCs) produced aggrecan (AGC) constantly during s econd and third weeks and TGFβ3 had no effect on it. In general, differentiated chondrocytes (D Cs) generated more AGC than NCs which was significantly different (P<0.001) in second week. In the pres ence of TGFβ3, DCs caused more significant production in s econd week but decreas ed in third week compared to other group without TGFβ3(P<0.001). (Figure 2, B) ( Table 2).
In general, type I collagen production increased i n all groups in third week in comparison to s econd week. However, articular chondrocytes (NCs) produced less type I collagen in two time points which showed significant difference (P<0. 05) only in third week. Adding TGFβ3 to D Cs and NCs yielded more type I collagen in third week indicating significant difference (P<0.05) compared to NCs and DCs without TGFβ3. (Figure 2, A) ( Table 2).
The results of GAG assay indicated that in third week articular chondrocytes (NCs) produced glycosaminoglycan (GAG) less than second week but the trend of its production in differentiated chondrocytes (DCs) was significantly increased in third week compared to second week (P<0.001). In addi tion, TGFβ3 resul ted in significant increase in GAG production than other groups without it in NCs and D Cs in two time points in that the highest amount was related to DCs (P<0.001). Generally, DCs generated more GAG than NCs in second and third weeks (Figure 3) ( Table 2).

Discussion
One of the aims of TE is to find a sui tabl e alternative cell source to synthesize an extra cellular matrix similar to natural cartilage (35). In recent years, several researchers have studied chondrogenesis in ADSCs and hav e compared them with other cells in different scaffolds and various growth factors (17)(18)(19)(20)(32)(33)(34)(35) but there is no detailed comparison between differentiated c ells from AD SCs and natural articular chondrocytes in alginate. In this study, we compared differentiated chondrocytes from ADSCs and articular chondrocytes separately in al ginate with and without TGFβ3 for 21 days.
According to our findings, the gene expression of type II collagen, GAG production and their progressive increas e during 21 days. In addition, the existence of aggrecan (AGC) in supernatant media as essenti al cartilage markers in differentiated chondrocytes (DCs), confirms that ADSCs hav e b een differentiated to chondrocytes. Comparing results of thes e markers between DCs and articular chondrocytes (NCs) after two weeks, indicated that gene expression of type II collagen was significantly lower than NCs, which was in consistent with previous reports about BM-M SCs in agarose and ADSCs in polyglycolic acid (PGA) (33,13). However, it was reported that in hyaluronic acid scaffold type II collagen expressed equally by ADSCs or chondrocytes and BM -MSCs expressed more than chondrocytes and the effect of BMP2 as well as TGFβ1 may have a role in this phenomenon. Unlike previous studies, our results indicated that DCs produced more AGC and GAG than NCs (33,32).Tigli et al (2009) indicated that embryonic stem cells (ESCs ), ESCs derived M SCs, ADSCs and BM-MSCs in silk and chitosan, expressed chondrogenic markers, such as type II collagen, Sox9 and AGC more than NCs (35) which is consistent with our findings about GAG and AGC but in contrast to type II collagen. This discrepancy may be due to the application of two growth factors, BMP2 and TGFβ1 at the same time and interaction between these factors and two other different scaffolds. Increasing level of GAG production in D Cs and superiority to NCs and also gene expression of type II collagen and generation of AGC in third week indicates that DCs in alginate during three weeks will maintain its phenotype. Decrease of some chondrogenic markers i n third compared to second week such as AGC in DCs or type II collagen and GAG production in NCs may be due to the fact that the highest amount of extracellular matrix was released on day14 and therefore the matrix molecules themselves caused reduction of thes e markers as a negative feedback and modification of chondrocyte metabolism (37,38). On the other hand, gene expression of type X collagen-a hypertrophic factor in embryonic stage-(39) was considered as a big challenge during differentiation of BM-MSCs and AD SCs to chondrocytes (30, 40,41) and chondrocytes redifferenti ation in various scaffolds and pellet culture (35,42,43). In this st udy, type X collagen in DCs was expressed significantly more than NCs after two weeks, but reduced considerably and distinguished after three weeks. Its concomitant with type II collagen gene expression and GAG production increase and continuation of AGC production at this point are positive signals for differentiation.
Of course the gene expression of type X collagen is not necessarily accompanied with collagen X protein production and hypertrophy (44) which may be the sign of rapid maturation of these cells (42). Some studies have reported that, healthy adult chondrocytes in tissue engineering hav e not-or very little-expressed this gene (45)(46)(47). Other studies stated that, articular chondrocytes expressed less type X collagen, compared to ESCs-derived MSCs in silk and chi tosan (35), ADSCs in hyaluronic acid (34) and pellet culture (48), which was in contras t with our present findi ngs in the third week. However, in line with our results, it was reported gene expression of type X collagen by BM-MSCs in silk and chitosan was lower than articular chondrocytes (35).
Types I and X collagens are considered as negative markers in chondrogenesis. It has been noted that MSCs especially ADSCs and BM -MSCs produce a lot of collagen type I (20,49,50). For TE, the expression of this gene is not appropriate and after in vivo implantation produces fibrocartilage instead of hyaline (13). Principally, scaffolds that maintain spherical shape of cells and prev ent their contact to each other -like agarose and algi nateenhance chondrogenesis and inhibit production of collagen type I (22). Progressive increase of type I collagen production in all groups of our s tudy during three weeks means that other factors may be involved other than scaffold and TGFβ3 in vitro. This gradual increase of ty pe I collagen is in line with the report of Jakobsen et al (34) but in contras t with Yang et al (51). In consistent wi th our findings, it was reported that type I collagen in MSCs in the pellet culture, silk and chitosan scaffolds was produced more than NCs (35,48). Conv ersely, it was reported that type I collagen in ADSCs and BM-MSCs in hyaluronic acid was equal to NCs (34).
The detection of AGC and type I collagen proteins in supernatant media by ELISA is due to lack of adequate structural consistency around the cells and washing away into the supernatant medi a. In a similar study, Jacobsen et al noted that despi te high gene expression of type II collagen by BM-MSCs encapsulated in hyaluronic, immunohistoc hemistry technique did not show a large quantity of type II collagen in resultant tissue, while it was detectable in supernatant medi a by ELISA (34).
Transforming growth factor type beta (TGFβ) is the frequently-used growth factor for chondrogenesis in vitro which has a major rol e in initiation of chondrogenesis. A lot of studies indicated i ts provoking role in proliferation and generation of cartilage matrix in progenitor cells (52, -54). The review of literature denoted that TGFβ has different effects in chondrogenesis. Other studies have mentioned that type II collagen and GAG production were stimulated by TGFβ (55) and s till some other groups have reported that it inhibits collagen and GAG production chondrocytes (56). TGFβ1 has a contribution in early stages (57) (20). Moreover, the improvement of synthesis of chondrogenic markers such as AGC, link proteins, fibromodulin and decori n in M SCs and pellet culture in the pres ence of TGFβ3 has been clarified (16,30). We found TGFβ3 improves chondrogenic markers such as AGC, GAG and type II collagen in D Cs, which is alignment with some other previous reports (33,55). Howev er; it i nhibits or has limited benefits in NCs which is in line with findings of Skantze et al (56) and in contras t with report of Mauck et al (33). Moreover, this growth factor has no effect in reducing of negative chondrogenic markers, types X and I collagens which is more prominent in third week. It was believed, TGFβ as a chondrogenic factor in vitro, causes retention of differentiated chondrocytes from MSCs in prehypertrophic state (60). Williams

Conclusion
ADSCs are multipotential cells easily obtainable from subcutaneous adipose tissue and so can be used as an alternative cell source for cartilage TE. According to our findings, despite low gene expression of type II collagen as a main chondrogenic marker, the trend gradually increased progressively from day 14 to 21. One reason is that more time may be required for ADSCs maturation after differentiation to chondrocytes compared to the natural articular chondrocytes (33).Type X collagen which is considered as a hypertrophic factor decreased during 21 days and possessed appropriate condition in DCs. However, type I collagen synthesis had a progressive trend that may lead to fibrocartilage instead of hyaline cartilage which is not suitable. It seems that more studies should be carried out to find new ways to produce type II collagen as much as possible and to inhibit type I collagen during chondrogenic differentiation of MSCs in various scaffolds in order to have efficient articular cartilage invitro to be used in clinics.